Fatty acids are main constituents of lipids, which are one of the three major nutrients for living organisms, and often refer to aliphatic monocarboxylic acids which are derived from natural lipids by hydrolysis. Generally, aliphatic chains of which are saturated are referred to as saturated fatty acids, and aliphatic chains of which contain a double bond or triple bond are referred to as unsaturated fatty acids. Fatty acids are classified into short-chain fatty acids (2 to 4 carbon atoms), medium-chain fatty acids (5 to 14 carbon atoms), long-chain fatty acids (16 to 18 carbon atoms), and very long-chain fatty acids (20 or more carbon atoms). When the number of carbon atoms is n and the number of double bonds is m, the fatty acids are often denoted by Cn:m.
The fatty acids are also the main constituents of the cell membrane of plants, and are important components accumulated predominantly in the form of triglycerides to provide energy sources in seeds and fruits. The amount of lipids accumulated in plants, and their fatty acid composition differ depending upon the types of plants. Examples of main fatty acids accumulated in plants include: palmitinic acid (C16:0) that is a saturated fatty acid with 16 carbon atoms (C16); and stearic acid (C18:0) that is a saturated fatty acid with 18 carbon atoms (C18). Other examples include unsaturated fatty acids with 18 carbon atoms (C18) having unsaturated bonds, such as oleic acid (C18:1) having one double bond, linoleic acid (C18:2) having two double bonds, and α-linolenic acid (C18:3α) having three double bonds. Plants containing a relatively large amount of these fatty acids, such as soybean, oil palm, sunflower, rapeseed, and coconut palm, are cultivated as fat or oil source plants (also referred to as oil plants). Note that, fatty acids having 18 or more carbon atoms and two or more unsaturated bonds (double bonds or triple bonds) are collectively referred to as Poly Unsaturated Fatty Acid (PUFA).
Incidentally, higher animals generally do not have desaturases involved in the syntheses of linoleic acid and α-linolenic acid, and therefore need intake of the PUFAs from plants (foods from vegetable sources). Therefore, linoleic acid and α-linolenic acid are referred to as essential fatty acids. In the body of higher animals, desaturation and elongation of carbon chains are repeated using these unsaturated fatty acids as substrates, so as to synthesize various unsaturated fatty acids, including dihomo-γ-linolenic acid, arachidonic acid (C20:4n-6), eicosapentaenoic acid (EPA) (C20:5n-3), and docosahexaenoic acid (DHA) (C22:6n-3).
It is known that these PUFAs have various functions for the metabolism in the body of higher animals, and play an important role as direct precursors of prostaglandins. Particularly, elderly people and infants, who have a reduced biosynthesis ability for dihomo-γ-linolenic acid, arachidonic acid, EPA, DHA, and fatty acids need intake of these fatty acids from foods. Particularly, arachidonic acid is known to be effective in improving senile dementia. Therefore, health foods mainly composed of arachidonic acid have been commercially available, and there has been an increasing demand for arachidonic acid.
Fish oil has a relatively high content of arachidonic acid, and arachidonic acid is now supplied in part by extraction from fish oil. However, in view of the problems such as depletion of fish, instable supply, and contamination of oil or fat resources caused by environmental pollution, arachidonic acid has been recently produced by microbial fermentation using microorganisms such as Mortierella, which is superior in terms of control of productivity, stability of long-term supply, cleanliness, and relative ease of purification, for example (e.g. see Document 1: Appl. Microbiol. Biotechnol., 31, p 11 (1987)). However, the microbial fermentation currently raises problems in that it requires a high production cost and a capital investment for scale-up, which cannot be carried out easily.
Therefore, if these PUFAs, particularly arachidonic acid, can be produced in oil plants, a significant improvement in the efficiency of their production can be expected, as well as cost reduction. In recent years, PUFA production in higher plants has been suggested by isolating desaturase genes and chain elongase genes, essential for the PUFA biosynthesis, from plants, animals, fungi, and yeasts, and by introducing these genes into higher plants.
Examples of plants whose oil or fat compositions are actually modified by genetic recombination include: (i) lauric acid-producing rapeseed (transgenic rapeseed obtained by isolating a medium-chain acyl-ACP thioesterase gene from laurel, which contains a relatively large amount of lauric acid, and then by introducing the gene, which specifically acts on C12:0-ACP (Acyl Carrier Protein) and releases lauric acid, into rapeseed by ligating it to the promoter of a napin gene that encodes a storage protein of the rapeseed; see Document 2: Science, 257, p 72 (1992)); (ii) high stearic acid content rapeseeds (recombinant rapeseeds with an increased stearic acid content as high as 40%, produced by introducing an antisense gene to suppress expression of a C18:0-ACP desaturase gene; see Document 3: Proc. Natl. Acad. Sci. U.S.A., 89, p 2624 (1992)); (iii) high erucic acid (C22:1) content rapeseeds (rapeseeds containing as high as 90% erucic acid, produced by introducing an LPAAT gene of yeast; see document 4: Plant Cell, 9, p 909 (1997)); (iv) high oleic acid content soybeans (soybeans with an increased oleic acid content as high as 80% compared with the original level of about 23%, produced by suppressing the expression of Δ12 desaturase gene Fad2 in soybean seeds and thereby suppressing the synthetic pathway producing linoleic acid from oleic acid, wherein a promoter derived from the β-conglycinin gene encoding a soybean seed storage protein was used as the Fad2-controlling promoter); and (v) γ-linolenic acid producing rapeseeds (rapeseeds produced by introducing Δ6 desaturase gene isolated from Borago officinalis; see Document 5: Proc. Natl. Acad. Sci. U.S.A., 94, p 4211 (1997)). Further, it has been reported that arachidonic acid and EPA were produced in flax plants by expressing Bacillariophyceae-derived Δ6 desaturase gene and Δ5 desaturase gene and a physcomitrella patens—derived chain elongase gene (see Document 6: J. Biol. Chem. 278, p 35115, (2003)).
Further, for the production of soybeans producing polyunsaturated fatty acids, gene introduction has been attempted by isolating the cDNAs of Δ6 desaturase, chain elongase, and Δ5 desaturase from Mortierella, which produces polyunsaturated fatty acids, and by ligating these cDNAs to various promoters (e.g. see document 11: “Shokubutu Riyou Enerugi Shiyou Gourika Seisan Gijutsu no Kenkyu Kaihatu Seika Houkokusho” (report on the results of research and development on biomass energy utilization rationalization industrial technology) reported in 2002; and document 12: Yoshikazu Tanaka, “Chikyu Shokuryou Shigen no tame no Shokubutu Baio Dai 160 Iinkai Dai 8 Kenkyukai Shiryou” (Material of 8th workshop in 160th Committee on biotechnology for global environment, foods, and resources), (Japan Society for the promotion of science), p 14-16, held on Jun. 13, 2003). Note that, the descriptions herein are based on Document 7: “Plant metabolic engineering”, NTS Inc., ISBN4-86043-004-2C3045, p 574-586 (2002), or document 8: J. Plant Physiol. 160, p 779 (2003), unless otherwise noted.
However, the description in Document 6 reporting on arachidonic acid-producing plants remains unclear and its disclosure is insufficient.
More specifically, for the introduction of foreign genes into plants to modify the composition or quality of oil or fat in the plants, it is necessary to control the expression of a gene of an enzyme involved in the determination of carbon-chain length, or a gene for a desaturase that determines the number and position of double bond. Further, for the production of fatty acids which are not inherent to the host plant, the time and site of fatty acid synthesis, and the form of the fatty acids in the cells must be considered to prevent adverse effects of the fatty acids on the growth of the host plant.
Still further, in the expression of genes of foreign organisms, particularly non-plants, there are cases where the transcripts are processed. In such a case, for example, codon modification or other process must be carried out (e.g. see document 9: Bio/Technology 11 p 194, 1993).
Further, enzymes involved in a series of biosynthesis reactions forms a complex in the cell, and metabolites of these enzymes may be metabolized through the molecular channel (e.g. document 10: Proc. Natl. Acad. Sci. U.S.A. 96, p 12929 (1999)). In such a case, even if a gene of an enzyme involved in the biosynthesis is known and its gene introduction technique is known, it is very difficult to predict how the enzyme produced by the introduced foreign gene functions and produces a desired substance in the host plant.
In this regard, Document 6 is insufficient because it is totally silent about such problems. As described above, the biosynthesis of fatty acid is unclear largely. Specifically, it is not clear as to whether (i) transcription and translation of fatty acid synthesizing genes derived from foreign organisms, e.g. Mortierella are carried out efficiently in plants, (ii) whether enzymes encoded by these genes can function well in plants, (iii) whether the enzymes can function cooperatively with a group of lipid synthetases in the cells of plants, or (iv) whether the arachidonic acid can accumulate in the form of triglycerides to provide an oil body as do other fatty acids, for example. That is, the production of arachidonic acid by the introduction of a foreign gene into plants takes tedious trial and error.
Further, as to legume plants, particularly soybeans, difficulties of genetic transformation by gene introduction have been pointed out, and there is scant information regarding transformation of soybeans. According to some reports, transformation efficiencies and regeneration efficiencies of soybeans are extremely low, and only some species of soybeans can be transformed (e.g. see Document 13: Santarem E R and Finer J J (1999), In Vitro Cell. Dev. Biol. Plant 35, p 451-455). Therefore, (i) it is necessary to develop a transformation system for soybeans, which do not easily accept foreign genes, and (ii) it is necessary to develop a stable multigene expression system which stably expresses multiple genes required for the synthesis of polyunsaturated fatty acids. In addition, (iii) it is necessary to confirm whether or not gene products derived from foreign organisms (enzymes involved in fatty acids synthesis) are actually expressed in the soybeans at a protein level and have an enzymatic activity, that is, whether or not lipid compositions of the transformed soybeans were altered.
Thus, the production of polyunsaturated fatty acids in soybeans is an extremely difficult technique and requires a multistage technological development. In fact, in the reports of Documents 11 and 12, transformant soybeans (plants) which produce polyunsaturated fatty acids are not obtained.
Further, in Documents 6, 11, and 12, there is no report on transformant plants whose trait of producing polyunsaturated fatty acids (e.g. arachidonic acid) is inherited to the next generation. That is, transformation of plants for the production of polyunsaturated fatty acids itself is attended with much technical difficulty. Therefore, it is much more difficult to obtain subsequent generations of plants that inherit the trait of producing polyunsaturated fatty acids.
Therefore, there is a strong demand for solving the foregoing problems and thereby realize, through trial and error, arachidonic acid-containing plants, particularly arachidonic acid-containing soybeans, which are produced by actually introducing a gene derived from foreign organisms into plants and then confirming not only its expression in a DNA level but also the expression of an enzyme in a protein level, followed by confirmation of the enzyme function. Further, it has been strongly demanded to obtain transformant plants that inherit the trait of producing polyunsaturated fatty acids to the next generation.